1. Field of the Invention
The present invention relates to an ethanol-tolerant yeast strain and uses thereof.
2. Description of the Related Art
Bioethanol production from plant or seaweed biomass has become the focus of world-wide concern with the long-term availability and deleterious environmental aspects of fossil fuels (Jeffries, T., and P. Lindbladm, 2009; Ragauskas, A. J., et al., 2006; Rubin, E. M., 2008). However, as one concern, the relatively high production cost of bioethanol has hindered investment in related industries. Much effort has been made to lower the costs of biomass procuration, pretreatment, fermentation, and product recovery (Xu, Q., A. Singh, and M. E. Himmel, 2009). During ethanol production, ethanol-producing microorganisms confront multiple stresses such as high initial substrate concentration, increased ethanol concentration, and accumulation of toxic byproducts. In addition to rapid growth and efficient fermentation capacity, the ability to tolerate these stresses is an important factor in choosing an ethanol producer (Ding, J., et al, 2009; Gibson, B. R., et al., 2007; Yoshikawa, K., et al., 2009). One way to improve ethanol yield is to obtain strains with enhanced stress tolerance.
Although not perfect, the yeast Saccharomyces cerevisiae has been diversely used as a primary microorganism for producing ethanol from biomass sources on an industrial scale. This organism is always exposed on various environmental stresses such as high-concentrated ethanol generated from an industrial ethanol fermentation process, leading to reduction of cell growth, cell viability and ethanol production (Casey and Ingledew, 1986). In this connection, there has been demanded the development of yeast strains to overcome stresses caused by high ethanol concentration. Furthermore, genome-wide analyses such as microarray and global expression pattern analysis have been utilized for identification of novel genes related to ethanol stress (Hirasawa, et al., 2007; Teixeira, et al., 2009; Yoshikawa, et al., 2009). Using these approaches, a variety of ethanol-tolerant genes have been identified as a non-essential gene. In addition, the results of previous reports in view of ethanol tolerance have been inconsistent with each other due to diverse strains and growth conditions (Teixeira, et al., 2009). Accordingly, it could be appreciated that a strain produced from the aforementioned genetic information is not always tolerant to ethanol stress condition (Yoshikawa, et al., 2009). Meanwhile, a deletion mutation library of commercially accessible Saccharomyces cerevisiae has been utilized for a genome-wide screening of ethanol-tolerant genes (Fujita, et al., 2006; Teixeira, et al., 2009; Yoshikawa, et al., 2009). Principally, previous studies isolated ethanol-sensitive mutants and genes thereof, followed by demonstrating a corresponding gene to be a gene for growth under high ethanol concentration condition.
In general, diverse genes have been known to affect cellular phenotypes (for example, severity of diseases, overexpression of metabolites, etc.) in a serious manner. Unfortunately, most cellular and metabolic engineering approaches have been performed by deletion or overexpression of single gene because of experimental limitations of vector construction and transformation efficiency. As a result, there have been excluded researches using mutations of several genes.
To investigate a mechanism to ethanol tolerance, numerous studies have been carried out. Especially, unsaturated fatty acid related to membrane fluidity was reported to be a critical factor of ethanol tolerance in yeasts (Kajiwara, et al., 2000; You, et al, 2003).
In addition, it has been reported that the accumulation of trehalose (Kim, et al., 1996) or proline (Takagi, et al., 2005) improves ethanol tolerance in yeasts, ergosterol is closely associated with ethanol tolerance of Saccharomyces cerevisiae (Inoue, et al., 2000).
In the mean time, VGH fermentation process has been generally utilized to obtain enormous amounts of ethanol during short fermentation, and has advantages as follows: (a) reduction of process steps; and (b) time and cost reduction. However, fermentation time was increased due to high glucose concentration, resulting in poor ethanol production. Consequently, the tolerance against both high ethanol and high osmosis caused by high glucose concentration in yeast is necessary to use VGH fermentation process
To develop ethanol-tolerant yeast strains, in addition to classic strategies such as evolutionary adaptation (Stanley, D., et al., 2010), random chemical mutagenesis (Mobini-Dehkordi, M., et al., 2008), and gene shuffling (Hou, L., 2010), three different approaches have recently been used: genome-wide DNA microarray analysis (Hirasawa, T., et al., 2007), transposon-mediated deletion mutant library (Takahashi, T., et al., 2001), screening of single gene knockout (SGKO) libraries (Auesukaree, C., et al., 2009; Fujita, K., et al., 2006; Kubota, S., et al., 2004; Teixeira, M. C., et al., 2009; van Voorst, F., et al., 2006; Yoshikawa, K., et al., 2009), and global transcriptional machinery engineering (gTME; Alper, H., et al., 2006). In the case of DNA microarrays, up- or down-regulated genes induced by ethanol stress are first identified as target genes and then their capability to confer ethanol tolerance is verified by overexpression for up-regulated genes or deletion for down-regulated genes. In the case of SGKO library screening, clones showing either diminished or enhanced growth are first isolated from screening in the presence of ethanol. Genes whose deletions cause slow growth are actually related with ethanol sensitivity and, therefore, should be verified for association with ethanol tolerance by overexpression. In contrast, genes whose deletions cause enhanced growth can directly used to construct ethanol-tolerant strains. However, the issue with these two approaches is that a huge number of target genes have been identified, representing as much as 5-10% of genes encoded in the yeast genome. Identification of ethanol-sensitive genes helps to understand the molecular basis of ethanol tolerance, but does not ensure the construction of ethanol-tolerant strains. Although it is easy and simple to prove whether overexpression of ethanol-sensitive genes confers ethanol resistance, few successful examples have been documented (Gibson, B. R., et al., 2007).
gTME reprograms the global transcriptional profile through random mutagenesis of one or more general transcriptional factors. This approach was first used to create a strain with enhanced ethanol tolerance by generating mutations of TATA-binding protein (TBP) encoded by SPT15, which could grow at a formerly lethal ethanol concentration (Alper, H., et al., 2006). However, other authors reported that this enhanced ethanol tolerance was not reproduced on a rich medium (Baerends, R. J., et al., 2009), which is not optional for industrial applications. Nevertheless, SPT15 mutations alter the transcription profile, presumably through the interaction with Spt3p, a subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex that regulates a number of RNA polymerase II-dependent genes. In addition, SPT15 mutations have been identified that were pleiotrophic (Eisenmann, D. M., et al., 1989) and some mutations in the regulatory domain of SPT15 resulted in transcriptional increase (Cang, Y., et al., 1999). These observations indicate that different mutations of SPT15 may induce expression of different sets of genes.
In this study, gTME was exploited as previously reported (Alper, H., et al., 2006) to create S. cerevisiae strains with ethanol tolerance. The present inventors obtained five ethanol tolerant strains (ETSs) containing different SPT15 mutant alleles and examined the effect of SPT15 mutations on ethanol tolerance. A genome-wide microarray was performed to identify genes related with ethanol tolerance and their functions were further examined using deletion mutants.
Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.